Integrating CRISPR technology with electrochemical sensing has promising potential in point-of-care testing applications. However, inappropriate immobilization of the reporter on the heterogeneous surface leads to a poor-cleavage efficiency. Additionally, the accuracy and reliability of electrochemical sensing still face challenges. Herein, an inverted tetrahedral DNA reporter was developed for electrochemical CRISPR aptasensing. Thiol-modified single-strand oligonucleotides were self-assembled on the edge of tetrahedral DNA nanostructures (TDNs) as a scaffold, enabling an inverted immobilization of DNA tetrahedra via the Au-S bond. The-cleavage activity of CRISPR/Cas12a on the single-stranded oligonucleotides resulted in the TDNs dissociating from the electrode surface. The recovery of electron transfer of potassium ferricyanide on the electrode enhances the electrochemical response, while the signal of adsorbed methylene blue on the skeleton of TDNs decreases, enabling a ratiometric signal output. As a proof of concept, the proposed inverted tetrahedral DNA reporters were employed to develop a label-free ratiometric electrochemical aptasensing method for kanamycin detection. Under the optimal conditions, as low as 0.35 pM kanamycin was detected in 50 min with a 4 orders of magnitude dynamic range from 1 pM to 10 nM. Furthermore, the practical application ability of the proposed method for kanamycin detection in a spiked milk sample was also demonstrated. This work offers a new perspective for electrochemical CRISPR sensing development. trans trans
